Cellular immune response to fractionated avian antigens by peripheral blood mononuclear cells from patients with pigeon breeder's disease

GINAL ATICLES Cellular immune response to fractionated avian antigens by peripheral blood mononuclear cells from patients with pigeon breeder's disease FELIPE MENDOZA, EMMA I. MELENDRO, MATILDE BALTAZARES,JOSI~ LUIS BANALES, CECILIA XIMI~NEZ, ROC[O CHAPELA, and MOISI~S SELMAN MEXICO CITY, MEXICO

Pigeon breeder's disease (PBD), a form of hypersensitivity pneumonitis caused by repeated inhalation of antigens of pigeon origin, is characterized by a diffuse inflammation of the lower respiratory tract. Although a variety of immunologic and nonimmunologic mechanisms have been described in the development of the disease, the pathogenesis is still far from clear. In this study we analyzed the T-lymphocyte proliferative response to a variety of avian antigens with use of peripheral blood mononuclear cells from 11 patients who had PBD and 10 healthy volunteers. We used a new method based on avian antigen- bearing nitrocellulose particles derived from Western blots to study the T-cell proliferative response to 15 antigenic fractions obtained from pigeon serum. With this technique, complex mixtures of antigens can be fractionated by polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and used for T-cell proliferation assays with selected antigenic determinants. A wide variety of responses were observed, and there were no reproducible patterns of reaction within either group. Nine of 10 healthy subjects responded to some soluble fractions. However, patients with PBD displayed the strongest response and responded to a significantly greater number of antigenic fractions. Fraction 2, representing a 220 kd molecular weight protein, was the only immunodominant antigen when both groups were compared; it was recognized by 73% of the patients with PBD and by only 20% of control subjects (p < 0.03). These findings show that T lymphocytes of patients with PBD recognize a wide range of bird proteins, which induce marked T-cell proliferation. (J LAB CklN MED 1996;127:23-8)

Abbreviations: EITB= enzyme immunotransfer blolCing; ELBA = enzyme-linked immunosorbent assay; HEPES- N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PBD = pigeon breeder's disease; PBS = phosphate buffered saline solution; PDE : pigeon dropping extracts; PSP = pigeon serum proteins; RPMI = (Roswell Park Memorial Institute) cell culture medium; SDSPAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SI = stimulation index

From the Instituto Nacional de Enfermedades Respiratorias, Facultad de Medicina UNAM, and Unidad Xochimilco UAM M6xico. Submitted for publication Jan. 12, 1995; revision submitted July 12, 1995; accepted Aug. 18, 1995. Reprint requests: Mois6s Selman, MD, Instituto Nacional de Enfermedades Respiratorias, Tlalpan 4502, Col. Secci6n XVI, CP. 14080, M6xico DF, M~xico.

Copyright © 1996 by Mosby-Year Book, Inc. 0022-2143/$5.00 + 0 5/1/68700

A n e d i t o r i a l r e l e v a n t to this a r t i c l e a p p e a r s o n p. 10 of this issue of the JOURNAL.

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Pigeon breeder's disease is the most common form of hypersensitivity pneumonitis in Mexico. 1 PBD is caused by repeated inhalation of antigens of pigeon origin and is characterized by a diffuse and predominantly mononuclear inflammation of the lower respiratory tract. 2 Although a variety of immunologic and nonimmunologic mechanisms have been described, the pathogenesis of PBD is still far from clear9 '4 The immunopathologic response involves both humoral and cellular mechanisms. However, whereas specific antibodies are present both in persons who have been exposed but have no symptoms and in patients with the disease, specific sensitized T lymphocytes and impaired suppressor T-cell function seem to occur more frequently in the patients with the disease. 5-7 On the other hand, it has been demonstrated by E I T B 8 and isoelectric focusing studies 9 that several epitopes from PSP and P D E are recognized by antibodies present in serum and bronchoalveolar lavage of patients with PBD. Nevertheless, studies concerning the cellular immune response to different fractions of the avian antigens are scanty. Recently, a technique has been described that uses the high resolving power of S D S - P A G E to separate antigens to assay the T-lymphocyte response. 1° This method has been successfully used in T-cell proliferation assays for antigen identification in tuberculosis and leprosy. 11'~2 The purpose of this study was to use this technique to analyze the lymphocyte proliferative response to fractioned pigeon serum antigen in patients with PBD. METHODS

Eleven nonsmoking patients with PBD (10 women and one man; mean age, 32 _+ 8 years) and 10 healthy nonsmoking volunteers were included in the study. Diagnosis was based on the following criteria: (1) a history of exposure to avian antigens predating the development of respiratory symptoms; (2) clinical symptoms and signs, abnormalities on chest x-ray films, and respiratory functional test results compatible with hypersensitivity pneumonitis; (3) the presence of serum antibodies against avian antigens; and (4) a lung biopsy (performed in all patients) with morphologic findings consistent with the diagnosis. 2"3 Briefly, the tissue samples showed diffuse interstitial inflammation of mononuclear predominance, mainly lymphocytes, and frequent multinucleated giant cells in terminal and respiratory bronchioles, as well as in the interalveolar walls. Foamy macrophages were seen in the alveolar spaces, and small and loosely arranged granulomas were observed in the interstitium. Biopsy cultures were negative for bacteria, mycobacteria, and fungi, and no changes suggestive of any other interstitial lung disease

J Lab Clin Med January 1996

were found. All patients were studied at the time of diagnosis and in the presence of active disease. None of the patients was being treated with immunosuppressive drugs at the time blood was collected. Control subjects (seven women and three men; 31 _+ 5 years of age) denied having contact with pigeons, and the search for specific antibodies against PSP by ELISA was negative. Avian antigens. A pool of 10 pigeon sera obtained by cardiac punction was used as the source of the antigens. Protein concentration of this pool, determined by Lowry's method, was 30 mg/ml. SDS-PAGE. Proteins from pigeon sera were separated according to the method described by Laemmli a3 with use of 10% polyacrylamide slab gels under nonreducing conditions. A mixture of standard proteins (Bio-Rad Laboratories, Hercules, Calif.) was used to estimate molecular weight. Samples of antigen contained 75 Ixg of protein per lane. Immunoblotting. Proteins resolved by SDS-PAGE were transferred to nitrocellulose sheets (LKB Diagnostics, Inc., Gaithersburg, Md.) with slight modifications as described by Towbin et al., 14 with use of 0.02 mol/L Trisglycine buffer, pH 8.3, containing 20% methanol, for 1 hour at 0.4 A. Blots were reversibly stained by dipping them for a few seconds in amido black (0.01% in 0.5% acetic acid), and they were washed with distilled water to permit visualization of transferred proteins. To eliminate amido black residue, blots were further washed with 0.2% Triton X-100 in 0.01 mol/L PBS, pH 7.4, for 1 hour and then with PBS alone for another hour. Nitrocellulose membranes were cut into 15 horizontal strips corresponding to 25 to 205 kd molecular weight fractions. Identical fractions of four different blots were pooled and treated together. Preparation of fractioned avian antigen bound to nitrocellulose. The 15 test fractions were fully dissolved by

incubation for 1 hour in 1 ml of dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.) at room temperature. The nitrocellulose particles with the adsorbed antigen were precipitated by gently adding 1 ml of sterile 0.05 mol/L carbonate-bicarbonate buffer, pH 9.6, under continuous vortexing. The samples were centrifuged at 10,000 g for 10 minutes, washed with RPMI 1640 medium (Sigma Chemical Co.), and finally resuspended in 2 ml of the same medium and stored at - 2 0 ° until used. In addition, the whole pigeon serum was also blotted in a nitrocellulose sheet under the same conditions as the fractions. Preparation of lymphocytes. Mononuclear cells obtained from peripheral blood mononuclear cells of normal subjects and patients with PBD were isolated by centrifugation on a discontinuous Ficoll-Hypaque density gradient and resuspended in RPMI 1640 medium supplemented with 10% AB + serum, 2 mmol/L L-glutamine, 10 mmol/L HEPES, 1 mmol/L pyruvate, 100 IU of penicillin/ ml, and 100 ixg streptomycin/ml (Gibco Laboratories, Gaithersburg, Md.). T-lymphocyte proliferation assay. A total of 2 × 105 peripheral blood mononuclear cells per well were cultured

J Lab Olin M e d V o l u m e 127, N u m b e r 1

in 96-well flat bottom microdilution plates (NUNC, Inc., Roskilde, Denmark). Serial dilutions (1:10, 1:20, 1:40, and 1:80) of the original antigen adsorbed onto nitrocellulose particles were used. Each dilution was added to each well in triplicate. As negative controls, protein-free nitrocellulose particles were used. The final volume of each well was adjusted to a concentration of 0.2 ml. To improve the cell-particle interaction, the plates were incubated at an angle of 45 degrees for 3 days and then horizontally for 4 additional days. Cells were cultured at 37° C in humidified air with 5% CO2. After 6 days of incubation, the cultures were pulsed with 1 ixCi of tritiated methylthymidine (6.70 Ci/mmol) (NEN, Research Product, Du Pont Co., Wilmington, Del.) for 18 hours and harvested onto glass fiber filters. The amount of tritiated thymidine incorporated was determined by liquid scintillation counting. At the end of the culture period, cell viability was assayed by trypan blue exclusion. Assays with viability greater than 85% are reported in this study. ELISA for serum antibody. Antibody activity in serum was determined according to a previously described ELISA. s Briefly, each well in a microtiter plate (NUNC, Roskilde, Denmark) was coated with 50 Ixl of a solution containing 2 ixg/ml of protein from pigeon serum in 0.1 mol/L carbonate buffer, pH 9.6. The plates were incubated overnight at 4° C and then washed twice with PBS containing 0.5% Tween 20 and blocked with 0.5% serum albumin in PBS Tween 20. The wells were next incubated for i hour at room temperature with human serum diluted 1:1500, washed, and further incubated with peroxidaseconjugated goat antihuman IgG 3,-chain specific (Cappel Laboratories, Organon Teknica Corp., Durham, N.C.) diluted 1:10,000. After washing excess conjugate, the plates were incubated for 10 minutes at room temperature with freshly prepared o-phenylenediamine and 3% hydrogen peroxide in citrate buffer, pH 4.5. Substrate conversion was assessed at 492 nm with use of a Microassay reader spectrophotometer (Diamedix Corp., Miami, Fla.), and antibody activity was obtained as the mean of triplicate optical density (0D492) readings. The sensitivity and specificity of the ELISA assay for avian antigen were 93% and 89%, respectively, as previously described. 8 EITB. To demonstrate the presence of avian immunoglobulins, a direct EITB was performed. Pigeon serum (75 fxg) was transfered onto nitrocellulose sheets by means of a transfer chamber (Bio-Rad Laboratories, Hercules, Calif.), according to the method described by Towbin. 14 The nitrocellulose sheets were blocked by incubating them overnight at room temperature in 0.02 mol/L PBS, pH 7.2, containing 1% bovine serum albumin. The blocked nitrocellulose sheets were then incubated with chicken anti-IgG (whole molecule; Sigma Chemical Co.) peroxidase conjugated in PBS bovine serum albumin for 60 minutes at room temperature. The sheets were washed twice with PBS Tween 20 and twice with PBS for 10 minutes each and then exposed for 3 to 5 minutes to the substrate solution, consisting of 3 mg of 4-chloro-l-naphtol/1 ml methanol (Sigma Chemical Co.) and 15 Ixl of 30%

M e n d o z a et al,

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Fig. 1. A, SDS-PAGE of pigeon serum. Molecular weights of markers are shown at left. B, A direct EITB of pigeon serum reacting against peroxidase-conjugated chicken anti-IgG. The 15 selected fractions for T-cell assay proliferation are indicated between panels A and B. Left arrow, 220 kd immunodominant fraction; right arrow, pigeon IgA.

H202 diluted in PBS, pH 7.2. Finally, the nitrocellulose sheets were thoroughly rinsed with water. Statistical analysis. The 1989 Epistat statistical computer program was used. Results are expressed as mean _+ SE. Results were compared with use of the U MannWhitney test and were considered significant when p was less than 0.05. Adjustment for multiple comparison was applied according to Bonferroni's procedure. RESULTS

W e first analyzed the quantitative difference in the response of patients and control T cells to whole blotted pigeon serum. Patients with P B D had an SI of 5.8 _+ 4.4, which was marginally significant w h e n c o m p a r e d with controls (2.1 _+ 0.6; p < 0.05). W e then randomly selected 15 fractions of the avian antigen (Fig. 1, lane A) corresponding to proteins from 25 to 225 kd molecular weight. The only criterion for this selection was the prominence of the bands. L y m p h o c y t e proliferation assays were p e r f o r m e d

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Mendoza

J L a b Clin M e d J a n u Q r y 1996

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Fig. 2. Proliferative responses of T lymphocytes from patients with PBD (upper panel) and control subjects (lower panel) to fractionated pigeon serum antigens. The SI was calculated as described in the text (see Results). The SI key is as follows: <5, white boxes; from 5 to 6.9, grey boxes; >7, black boxes. OD, Optical density for specific avian antigens antibodies as determined by ELISA.

separately with the 15 antigen fractions obtained from the nitrocellulose blot. Initially, four dilutions from each fraction were used. After several preliminary assays it was demonstrated that 1:10 and 1:20 dilutions were useful, and these dilutions were subsequently used. To compare profiles of different donors, the results were expressed as the SI ratio between the mean counts per minute obtained from cultures stimulated with test fractions and the mean counts per minute obtained from cultures with negative controls. In the patients with PBD, several fractions usually induced a strong proliferative response of lymphocytes. By contrast, in control subjects an SI score higher than 7 was only occasionally observed. Fig. 2 shows the results of all tested patients and control subjects. A wide variety of responses were observed, and there was no distinctive pattern of reaction within either group. Nine of 10 healthy subjects responded to some fractions. However, most patients with PBD responded to more fractions and

with a higher SI score than control subjects. In general, high molecular weight fractions induced higher proliferative responses. Especially notable is fraction 2, of approximately 220 kd, which induced a positive response in 73% of the patients with PBD and stimulated only a weak response in 20% of control subjects. To find out if this 220 kd antigen is related to the pigeon IgA, we made an enzyme immunotransfer with chicken anti-IgG, which presents cross-reaction with pigeon IgG and IgA. This immunoglobulin was observed at the same level of the immunodominant fraction (Fig. 1, lane B). The mean Sis to each fraction for both patients with PBD and controls are shown in Fig. 3. Fraction 2 was the only immunodominant antigen when the two groups were compared (p < 0.05). On the other hand, we also analyzed the number of antigens that induced a proliferative response in control subjects and patients with PBD. Fifty percent of control subjects recognized only one antigen, and only one

J Lab Clin M e d V o l u m e 127, N u m b e r 1

M e n d o z a et ol.

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Fig. 3. SI response of T cellsfrompatientswithPBD and controlsubjectsto the 15 fractionatedantigens (mean + standard error). Fraction2 was the onlyimmunodominantantigen (p < 0.05). healthy volunteer recognized five bands. In contrast, 50% of the patients with PBD had a positive response to five or more fractions. Interestingly, there was no relationship between the SI and the levels of specific antibodies against pigeon serum measured by ELISA. DISCUSSION

The mechanisms involved in the pathogenesis of PBD appear to be rather more complex than formerly suspected. Immune-complex formation and complement activation might play a role during the early inflammatory phase of the disease. However, much evidence supports a more important role of cellular immunity than humoral hyperresponsiveness in the development of the alveolitis. 2-4 The conspicuous histopathologic feature is diffuse mononuclear cell inflammation, which may organize into granulomas. Bronchoalveolar lavage has demonstrated that inflammation is mainly mediated by T cells, with an increase of the CD8 ÷ suppressor/cytotoxic phenotype) 5 Likewise, sensitized lymphocytes produce lymphokines after stimulation with avian antigens. 6'15'16 In addition, the disease has been experimentally reproduced in nonsensitized animals by transferring T cells from sensitized animals, and it has not been reproduced with immunoglobulins. 17'18 Moreover, minimal lung lesions are produced in athymic nude mice exposed to repeated administration of antigens. 19 It is important to note that until now most studies concerning the immune response in PBD have used

the complete serum or crude extracts from pigeon droppings as avian antigens. Both are formed by a complex mixture of bird proteins, and high and low molecular weight proteins carry reactive epitopes. 8 It has been reported that pigeon droppings contain proteins identical to, as well as cross-reactive with, PSP. Therefore it is feasible that inhalation of PDE during the handling of pigeons or the cleaning of the excretions may lead to immunization, resulting in the production of antibodies against PSP. However, which component(s) of this mixture is responsible for a given immune response is not yet known. Improved characterization of the cellular immune response to different pigeon antigens may be important for the study of immunopathogenesis of PBD. Our results show great heterogeneity in response to fractions both in healthy subjects and patients with PBD. Because we know that T cells recognize antigens bound to molecules of the major histocompatibility complex, the recognition of different components might be related to the polymorphism of the major histocompatibility complex gene products, as suggested for other diseases. 2°'21 Half of the healthy control subjects displayed a significant, albeit low, SI response to at least one antigen fraction, although the fractions they responded to were usually different. This finding is not surprising because serum from healthy persons has been reported to react positively by immunodiffusion and immunoelectrophoresis with several components of pigeon antigens. 22 However, patients responded to a larger number of antigenic fractions

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Mondoza et al.

with higher SI scores than did control subjects. This finding supports the notion that several antigens of different molecular weight contribute to the overall polyclonal T-cell response to pigeon serum antigens. In spite of heterogeneity, a high molecular weight (220 kd) immunodominant antigenic fraction was recognized by more than 70% of patients with PBD and by only 20% of control subjects. The lack of response to this antigen observed in three patients may be the result of a predominant local lung response. In this sense, Moore et al. 6 have found that in some patients with PBD, T lymphocytes from bronchoalveolar lavage were stimulated by avian antigens, whereas peripheral T cells were not stimulated. The nature of this immunodominant fraction is presently unknown, but interestingly this antigen reacted with antibodies against chicken anti-IgG, which have a cross-reaction with pigeon IgA and IgG. This finding suggests that this fraction may contain or may be the IgA, as previously insinuatedY In the present study there was no relationship between the antibody levels and either the intensity of the cellular immune response or the number of stimulatory fractions. These results are not unexpected, because the same results have been observed with other diseases such as tuberculosis or collagen-vascular diseases. 24 It is therefore relevant to study both the cellular and the antibody immune response. Even though we found one immunodominant fraction in pigeon serum antigen; we cannot disregard the possibility that PDE antigens may contain some other important antigens. The biologic function of this immunodominant antigen is presently unknown. However, this fraction might play a role in the pathogenesis and diagnosis of the disease. Further studies are necessary to determine if this,~antigen represents a major pathogenic component of the T-cell repertoire reactive with pigeon proteins. REFERENCES

1. P6rez-Padilla R, Salas J, Chapela R, et al. Mortality in Mexican patients with chronic pigeon breeder's lung compared with those with usual interstitial pneumonia. Am Rev Respir Dis 1993;148:49-53. 2. Selman M, Chapela R, Salas J, et al. Hypersensitivity pneumonitis: clinical approach and an integral concept about its pathogenesis--a Mexican point of view. In: Selman M, Barrios R, eds. Interstitial pulmonary diseases: selected topics. Boca Raton, Fla.: CRC Press, 1991:171-96. 3. Selman M, Chapela R, Raghu G. Hypersensitivity pneumonitis: clinical manifestations, pathogenesis, diagnosis, and therapeutic strategies. Semin Respir Med 1993;14:353:64. 4. Salvaggio JE. Recent advances in pathogenesis of allergic alveolitis. Clin Exp Allergy 1990;20:137-44. 5. McSharry C, Banham SW, Lynch PP, Boyd G. Antibody

6.

7.

8.

9.

10.

11.

12.

13. 14.

15. 16.

17.

18.

19.

20.

21.

22. 23. 24.

measurement in extrinsic allergic alveolitis. Eur J Respir Dis 1984;65:259-65. Moore VL, Pedersen GM, Hauser WC, Fink JN. A study of lung lavage materials in patients with hypersensitivity pneumonitis: in vitro response to mitogen and antigen in pigeon breeder's disease. J Allergy Clin Immunol 1980;65:365-70. Keller RH, Fink JN, Lyman S, Pedersen G. Immunoregulation in hypersensitivity pneumonitis: I--differences in T-cell and macrophages suppressor activity in symptomatic and asymptomatic pigeon breeders. J Clin Immunol 1982;2:46-52. Sandoval J, Baflales JL, CortEs JJ, Mendoza F, Selman M, Reyes P. Detection of antibodies against avian antigens in bronchoalveolar lavage from patients with pigeon breeder's disease: usefulness of enzyme-linked immunosorbent assay and enzyme immunotransfer blotting. J Clin Lab Anal 1990;4:81-85. McCormick DJ, Fredricks WW, Tebo TH, Calvanico NJ. The antigens of pigeon breeder's disease: VII--isoelectric focusing studies on unfractionated pigeon dropping extract. Immunology 1982;129:1493-8. Lamb JR, O'Hehir RE, Young DB. The use of nitrocellulose immunoblots for the analysis of antigen recognition by T lymphocytes. J Immunol Methods 1988;110:1-10. Lamb JR, Young DB. A novel approach to the identification of T-cell epitopes in Mycobacterium tuberculosis using human T-lymphocyte clones. Immunology 1987;60:1-5. Filley E, Abou-Zeid C, Waters M, Rook G. The use of antigen-bearing nitrocellulose particles derived from western blots to study proliferative responses to 27 antigenic fractions from Mycobacterium leprae in patients and controls. Immunology 1989;67:75-80. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;277:680-2. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrilamyde gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350-4. Costabel U. The alveolitis of hypersensitivity pneumonitis. Eur Respir J 1988;1:5-9. Schuyler MR, Thigpen TP, Salvaggio JE. Local pulmonary immunity in pigeon breeder's disease. Ann Intern Med 1978; 88:355-8. Schuyler M, Subramanyan S, Hassan MO. Experimental hypersensitivity pnumonitis: transfer with cultured cells. J LAB CLIN MED 1987;109:623-30. Schuyler M, Gott K, Sopp G, Crooks L. CD3+ and CD4+ cells adaptively transfer experimental hypersensitivity pneumonitis. Am Rev Respir Dis 1992;146:1582-8. Takisawa H, Ohta K, Horiuchi T, et al. Hypersensitivity pneumonitis in athymic nude mice: additional evidence of T cell dependency. Am Rev Respir Dis 1992;146:479-84. Mendez PS, Lamb J, Bothamley G, Stanley P, Ellis C, Ivanyi J. Molecular study of the T cell repertoire in family contacts and patients with leprosy. J Immunol 1989;142:3599-604. Lamb JR, Lathgra R, Rothbard JB, et al. Identification of mycobacterial antigens recognized by T-lymphocytes. Rev Infect Dis 1989;11:$443-7. Tebo TH, Moore VL, Fink JN. Antigens in pigeon breeder's disease. Clin Allergy 1977;7:103-8. Fredericks W. Antigens in pigeon dropping extracts. J Allergy Clin Immunol 1978;61:221-3. Pham BN, Prin L, Gosset D, et al. T lymphocyte activation in systemic lupus erythematosus analyzed by proliferative response to nucleoplasmic proteins on nitrocellulose immunoblots. Clin Exp Immunol 1989;77:168-74.